Eukaryotic
genes are interrupted by stretches of
noncoding DNA sequence, which are removed from thenewly-synthesized
pre-messenger RNA to ensure accurate expression of genetic information.
In most higher-eukaryotic organisms, such as humans, these noncoding
sequences, introns, interrupt the majority of genes, and can be up to
100,000 bases. Hence, intron recognition is an integral step in gene
expression.

Introns are
excised from pre-messenger RNAs by a
large ribonucleoproteincomplex called the spliceosome, which is
comprised of 5 small nuclear RNAs and a large collection of protein
factors. The spliceosome is well-conserved from yeast to humans, and it
undergoes dramatic, ATP-dependent rearrangements to allow for multiple,
ordered intron recognition events and splicing catalysis. Two
fundamental challenges to understanding the mechanism of pre-mRNA
splicing are to characterize the dynamic RNA-RNA rearrangements that
are critical for establishing the catalytic center of the spliceosome
and to determine the roles of the numerous splicing proteins that are
involved in this process.

Each
of the steps in synthesis and processing
of a messenger RNA (including pre-messenger RNA splicing) have been
studied as distinct biochemical reactions. Nevertheless, there is
growing evidence that in vivo, these reactions are spatially and
temporally coordinated. The splicing machinery appears to associate
with the pre-messenger RNA co-transcriptionally, and the transcription
apparatus, including the RNA polymerase (specifically the
hyperphosphorylated C-terminal domain, or CTD, of the RNA polymerase
II), helps to recruit splicing factors to the nascent RNA
transcript. In order to understand these
critical events in eukaryotic gene expression, we are exploiting the
power of yeast genetics and biochemistry using the model organism
Saccharomyces cerevisiae. S. cerevisiae is not only experimentally
tractable, but its splicing machinery is very similar to that of
mammals , and the genes encoding most of the splicing factors have been
identified.

(1) Characterization of
RNA-RNA and RNA-protein interactions involved
in splice site recognition. One of
goals of the lab is to understand the dynamic
rearrangements carried out by the spliceosome. In particular we have
focused on the RNA-RNA and RNA-protein interactions that mediate 5'
splice site recognition using a trans-splicing/crosslinking system. In
vitro splicing reactions are carried out using pre-mRNAs in which the
5' splice site is contained on an RNA substituted with photoreactive,
nucleoside triphosphate analogs at positions around the 5' splice site,
while the 3' splice site/branchpoint are contained on a separate
molecule. In this trans-splicing system, splicing proceeds through both
catalytic steps to generate an accurately spliced product. Upon
UV-irradiation, crosslinking is induced between the pre-mRNA substrate
and the small nuclear RNAs or proteins in the reaction, allowing us to
"freeze" interactions that occur between the pre-mRNA and components of
the spliceosome during pre-mRNA splicing. Using this system, we have
identified a number of novel interactions that take place during splice
site recognition. Further, using extracts derived from yeast strains
with mutations in specific splicing proteins (including the RNA
helicases required for splicing), we have been able to block splicing
at discreet steps along the splicing pathway, identify which proteins
are responsible for mediating the observed crosslinks, and are
constructing a temporal map of the interactions that occur during
splicing.

(2) Identification and
characterization of functional interactions
between the splicing and transcription machineries. With
the growing appreciation of the close
spatial and temporal relationship between transcription and splicing,
it is clear that interactions between the transcription machinery and
the splicing machinery play an important role in the two reactions.
This has led to a model in which the transcription machinery plays a
role in facilitating pre-mRNA splicing and the splicing machinery and
the splicing reaction can alter the transcription properties of the
polymerase (Figure 1). To explore this model, we have initiated a
three-pronged approach. First we are carrying out a genetic analysis to
identify specific factors that act at the interface of transcription
and splicing. We are complementing this approach by using biochemical
tools, including affinity purification and co-immunoprecipitation, to
characterize physical interactions between these factors. Finally, we
are employing a combination of in vivo and in vitro splicing and
transcription assays to elucidate the mechanisms by which splicing and
transcription are coordinated, including the mechanism by which
pre-mRNA splicing occurs within the context of mRNA synthesis from a
chromatin template.

Figure 1.
Co-transcriptional splicing

Dr. Tracy Johnson received her
Ph.D. from the University of California,
Berkeley and was a Jane Coffin Childs postdoctoral fellow at the
California Institute of Technology.